The long-term goal of this project is to elucidate the cellular mechanisms of parathyroid hormone receptor (PTH1R) signaling and action. Recent advances reveal how cytoplasmic adapter proteins regulate PTH1R effects. Our studies show that amino-truncated PTH fragments promote receptor endocytosis without concomitant activation. One such adapter protein, NHERF1, governs these effects. Thus, activation and inactivation of the PTH1R can be dissociated in a cell- and ligand-specific manner. These findings suggest that the PTH1R may adopt multiple conformations and not exist simply in "on" or "off" states. Preliminary studies revealed that additional adapter proteins contribute to the ligand- and cell-specific signaling and trafficking of the PTH1R. The proposed studies use an array of complementary biophysical, molecular and biochemical, and cell biological tools to address the unifying hypothesis that PTH1R function is dictated by conformation and its interactions with targeted binding partners. Four specific aims are developed to test this hypothesis. Aim 1 will characterize dynamic PTH1R conformational changes. This will be accomplished by applying time-resolved FRET and single molecule TIRF techniques. Wild-type PTH1Rs, as well as receptors harboring known activating and inactivating mutations, or conformationally restrained modifications, will be studied. Aim 2 defines the signaling and trafficking of PTH1R isoforms. Preliminary studies describe alternate donor and acceptor splice sites at the exon 14/15 junction. We hypothesize that PTH1R(e14 results in a misfolded receptor that traffics and signals inefficiently, and acts as a functional dominant negative of the wild-type PTH1R. A combination of biophysical fluorescence techniques combined with molecular and cell biological approaches will be applied to test this theory. Aim 3 will delineate PTH1R interactions with Dishevelled (Dvl) that regulate canonical (-catenin signaling. Our preliminary work identified a Dvl-binding motif in the proximal tail of the PTH1R that mediates the effects of PTH on (-catenin activation. These preliminary studies will be pursued to test the hypothesis that Dvl and NHERF1 act as a molecular router to regulate convergent cell- specific PTH1R effects on Wnt/(-catenin signaling. Aim 4 describes the actions of small molecules that disrupt PTH1R PDZ-recognition and Dvl domains on the biological actions of PTH. Innovative designer molecules based on molecular modeling will be used to test the hypothesis that unique small molecules selectively regulate PTH1R signaling and trafficking. These studies will quantitatively examine the relations between PTH1R structure and function and characterize a novel mechanism to explain the regulation of PTH1R effects. The studies will generate new information that is relevant to understanding bone turnover. The outcomes will help define small molecules and potential therapeutic targets for improved treatment of osteoporosis and other metabolic bone diseases. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to test how parathyroid hormone and (-catenin pathways, which have been shown separately to regulate bone formation and turnover, do so through an interacting mechanism. The outcome of the experiments is highly relevant to our understanding of osteoporosis and will define potential therapeutic targets for improved treatment of osteoporosis and other metabolic bone diseases.